Because of the very low level of HCV present in the serum of infected individuals, as well as the low level of replication in the host, reverse transcription-polymerase chain reaction (RT-PCR) assays are the only method suitable for the routine detection of HCV RNA. The use of RT-PCR to monitor HCV replication in vivo as well as the in vitro inoculation of cultured cells presents unique problems. The extreme sensitivity of PCR permits the detection of HCV RNA in tissues not permissive for replication of the virus, and following in vitro infections, the residual inoculum can be detected for extended time periods depending on the sensitivity of the PCR procedure. Since HCV is a positive-stranded RNA virus, the detection of negative-strand RNA should be indicative of active viral RNA replication, assuming that the inoculum contains primarily positive-strand RNA. Early attempts to detect negative-strand RNA employed a strand-specific PCR technique that utilized only one primer during cDNA synthesis, followed by inactivation of the RT and amplification of the cDNA by PCR. During the course of our studies, we found this technique to lack significant strand specificity using synthetic RNA. A series of experiments suggested that the lack of specificity was probably owing to a combination of factors, including false priming of the incorrect strand (e.g., the positive strand in a negative-strand assay) by the cDNA primer, self-priming of the RNA, and random priming by extraneous nucleic acids (illustrated in Fig. 1 A). Fig. 1 Schematic diagram of conventional strand-specific RT-PCR and rTth RT-PCR procedures. (A) The amplification of HCV negative-strand RNA using conventional strand-specific RT-PCR and the false amplification of the HCV positive-strand RNA by the same procedure A cDNA copy of the RNA is made with primer complementary to negative-strand RNA. At the reduced temperatures used for cDNA synthesis, this primer can misanneal at sites on the positive-strand RNA with partial homology to the primer. cDNA synthesis can also be primed by contaminating nucleic acids and by terminal hairpin structures that can occur anywhere in genome if the RNA is partially degraded Following cDNA synthesis, the RT activity is inactivated and PCR amplification is performed with Taq. False-primed positive-strand RNA yields the same product as negative-strand RNA provided that the cDNA produce from false priming spans the sequence encompassed by the PCR primers. (B) The amplification of HCV negative-strand RNA by rTth RT-PCR is depicted A cDNA copy of the RNA is made with a primer complementary to the negative strand of RNA using the rTth thermostable reverse transcriptase at 70°C The RT activity of rTth is inactivated by chelation of Mn(2+) with EGTA. Following the addition of Mg(2+) and the reverse primer, PCR is conducted using the thermostable DNA polymerase activity of rTth.